The invention relates to a method for the activation of a Diels-Alder reaction. More specifically, the invention relates to the activation of a Diels-Alder reaction in a sterol 5,7-diene.
In certain processes for the commercial production of sterols, a Diels-Alder addition is useful as a protection step and also for purification. Diels-Alder addition has proven useful in the commercial production of 25-hydroxyvitamin D3. With the present state of the art, the economics of commercial 25-hydroxyvitamin D3 production is strongly affected by the yield of the Diels-Alder reaction. It is therefore desirable to maximize the yield of the Diels-Alder reaction in commercial sterol production.
Diels-Alder reactions are the well-known chemical addition of a dienophile to a diene. Textbook methods to activate Diels-Alder reactions use electron-releasing groups to enhance the electron density of the pi-donor diene. Similarly, electron-withdrawing groups are used to decrease the electron density of the pi-acceptor, thus enhancing its electrophilicity. In some processes, such as commercial sterol production, it is not possible or practical to provide functional groups to activate the electron donor for a Diels-Alder reaction. Means to enhance a Diels-Alder reaction in commercial sterol production is sought.
In the art, a semisynthetic commercial production of 25-hydroxyvitamin D3 involves the saponification of 5,7,24-cholestatrienyl esters from fermentation of a double mutant yeast to form the free 5,7,24-cholestatrienol (also known as 7-dehydrocholesterol or provitamin D3). The mutant yeast here contains the erg6 mutation in zymosterol-24-methyltransferase, and a mutation in the expression of ergosta-5,7,24(28)-trienol-22-dehydrogenase enzyme (erg5) as described in U.S. Pat. No. 5,460,949, whose disclosures are incorporated by reference.
Saponification procedures are common in the art, particularly where the hydrolysis of esters in organic matter is desired. Saponification reactions involve treatment with a strong base, typically with heating.
The saponification reaction product also includes squalene, fatty acids, including saturated and unsaturated fatty acids, and other sterols, which can include unconjugated dienols, and mono-unsaturated sterols. To separate the cholestatrienol and other sterols including lanosterol, 4,4,-dimethylzymosterol, 4-methylzymosterol, zymosterol, cholesta-7,24-diene-3xcex2-ol from that mixture. The pH is typically adjusted to about 7-8 pH units, and the saponificate is extracted with heptane, and the heptane extract is washed with water. The washed heptane extract is concentrated, and mixed with ethyl acetate. This is called the xe2x80x9csterol extractxe2x80x9d.
In a Diels-Alder protection and/or purification step of the art, the sterol extract is reacted with a dienophile to form a Diels-Alder adduct of the 5,7-diene of the cholestratrienol, while leaving the unconjugated dienols and other sterols unreacted. The dienophile is typically phthalhydrazine generated in situ from the reaction of aqueous bleach (sodium hypochlorite) on phthalhydrazide. The Diels-Alder adduct is then chromatographically separated from the unreacted yeast sterol mixture, the cholestatrienol is regenerated, further purified and used to make 25-hydroxyvitamin D3. This reaction sequence is illustrated in U.S. Pat. No. 5,391,777, whose disclosures are incorporated by reference.
U.S. Pat. No. 5,208,152 describes catalysts of Diels-Alder reactions where the substrates are cyclic conjugated dienes having a fugitive leaving group. Those dienes are unlike sterols.
U.S. Pat. No. 4,503,195 discloses the use of di- and tri-phenylated cation radical polymers as Diels-Alder catalysts, wherein the cation radical is a Group VA element (e.g. nitrogen, phosphorus or arsenic).
U.S. Pat. No. 4,413,154 and U.S. Pat. No. 4,384,153 disclose Diels-Alder reactions of 1,3-butadiene and 4-vinylcyclohexene over molecular sieves/zeolites.
In accordance with the present invention, it has unexpectedly been found that the presence of at least a catalytic amount of an unsaturated C12-C24 fatty carboxylic acid enhances the formation of Diels-Alder adduct between a steroid 5,7-diene and a dienophile (preferably generated in situ from an oxidizable dienophile precursor and an oxidant). According to the invention, at least a catalytic amount of an unsaturated C12-C24 fatty carboxylic acid is exogenously provided to a Diels-Alder reaction.
The steroid 5,7-diene can be any such compound. Those that have been examined all undergo the Diels-Alder reaction with enhanced yield of adduct when at least a catalytic amount of a C12-C24 unsaturated carboxylic acid is added. Ergosterol and 5,7,24-cholestatrienol are preferred steroid 5,7-dienes, with 5,7,24-cholestatrienol being particularly preferred.
The present invention also contemplates an improved method of forming a Diels-Alder adduct with a 5,7-diene sterol comprising the following steps. A 5,7-diene sterol is admixed with a dienophile to form a reaction mixture, and thereby converting the 5,7-diene sterol to a Diels-Alder adduct. The 5,7-diene sterol has the structural formula 
wherein R3 is selected from the group consisting of H and R1COxe2x80x94 wherein R1 is monocyclic aryl of 5 to 7 carbon atoms or lower alkyl, and R4, R5 and R6 are independently selected from the group consisting of H, hydroxyl, and lower alkyl, and R7 is a C1 to C10 hydrocarbyl group. In one preferred embodiment, the 5,7-diene steroid compound is cholesta-5,7,24-triene-3-ol. Further chemical conversion of the Diels-Alder adduct to provide a modified Diels-Alder adduct is optionally conducted. The Diels-Alder adduct or the modified Diels-Alder adduct is separated from the mixture.
The invention contemplates adding at least a catalytic amount of an ethylenically unsaturated C12 to C24 fatty acid to the Diels-Alder reaction mixture. Preferably, the ethylenically unsaturated C12 to C24 fatty acid is linolenic acid, linoleic acid, oleic acid or a mixture of two or all three acids.
In one embodiment, the dienophile has the structural formula Xxe2x80x94Rxe2x95x90Rxe2x80x94Y wherein the R groups are both N or both C-Q where the Q groups are H or together form a third bond, and wherein X and Y are selected from electron-withdrawing groups themselves independently selected from the group consisting of xe2x80x94COOH, xe2x80x94CHO, xe2x80x94NO2, xe2x80x94CN, xe2x80x94COOR8 and xe2x80x94COR8, where R8 is lower alkyl, or wherein X and Y are linked together to form a xe2x80x94(CO)xe2x80x94Zxe2x80x94(CO)xe2x80x94 bridge in which Z is lower alkylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, or xe2x80x94NR2 wherein R2 is lower alkyl, H or monocyclic aryl of 5 to 7 carbon atoms and up to 5 ring substituents, wherein the ring substituents are selected from the group consisting of xe2x80x94(CH2)nxe2x80x94NH2, xe2x80x94(CH2)nxe2x80x94COOH, xe2x80x94NO2, halogen and lower alkyl, where n is an integer that is zero to 6, inclusive.
In another embodiment, the dienophile is generated in situ from (i) an oxidizable dienophile precursor and (ii) an oxidizing agent effective to oxidize the precursor to form the dienophile, wherein the dienophile precursor has the structural formula Xxe2x80x94NHxe2x80x94NHxe2x80x94Y wherein X and Y are selected from electron-withdrawing groups themselves independently selected from the group consisting of xe2x80x94COOH, xe2x80x94CHO, xe2x80x94NO2, xe2x80x94CN, xe2x80x94COOR8 and xe2x80x94COR8 where R8 is lower alkyl, or wherein X and Y are linked together to form a xe2x80x94(CO)xe2x80x94Zxe2x80x94(CO)xe2x80x94 bridge in which Z is lower alkylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, or xe2x80x94NR2 wherein R2 is lower alkyl, H or monocyclic aryl of 5 to 7 carbon atoms and up to 5 ring substituents, wherein the ring substituents are selected from the group consisting of lower alkyl, halogen, xe2x80x94NO2, xe2x80x94(CH2)nxe2x80x94NH2, and xe2x80x94(CH2)nxe2x80x94COOH, where n is an integer that is zero to 6, inclusive. Preferably, the oxidizable dienophile precursor is phthalhydrazine. Preferably, the oxidizing agent is hypochlorite (e.g. NaOCl or an aqueous solution of sodium hypochloride).
The present invention further contemplates a Diels-Alder reaction mixture initially comprising at least a catalytic amount of an exogenously added ethylenically unsaturated C12 to C24 fatty acid, a steroid 5,7-diene and a dienophile as discussed above.
The present invention has many benefits and advantages, several of which are listed below.
One benefit of the invention is that the Diels-Alder adduct is formed in enhanced yield.
An advantage of the invention is that the catalyst is quite inexpensive compared to the cost of the steroid co-reactant.
Another benefit of the invention is that the enhancement of Diels-Alder adduct formation leads to protection for a larger proportion of the 5,7-diene-containing sterol already present when the Diels-Alder addition is used for protection of the conjugated diene functionality.
Another advantage of the invention is that the enhancement of Diels-Alder adduct formation leads to more easy separation for a larger proportion of the 5,7-diene-containing sterol already present when the Diels-Alder reaction is used to assist in purification.
A further benefit of the invention is that neutralization of a saponification reaction with acid is not needed to carry endogenous unsaturated fatty acid forth in semisynthetic sterol production.
Still further benefits and advantages will be apparent to the worker of ordinary skill from the disclosure that follows.
The present invention is, in part, an improvement on the method of U.S. Pat. No. 5,391,777, the disclosures of which are incorporated herein by reference. U.S. Pat. No. 5,391,777 describes the use of a Diels-Alder reaction to separate cholesta-5,7-diene-3xcex2-25-diol and other steroid 5,7-dienes from a complex sterol solution. A 5,7-diene-containing steroid is reacted with a dienophile or an oxidizable dienophile precursor in combination with an oxidizing agent to provide a Diels-Alder adduct of the diene.
In the specification and the claims that follow, reference is made to a number of terms that are defined as follows.
xe2x80x9cExogenousxe2x80x9d or xe2x80x9cexogenously addedxe2x80x9d used herein in reference to a suitable unsaturated fatty acid that is separately added to the 5,7-diene-containing sterol, rather than already being with the sterol. For example, this does not refer to unsaturated fatty acid present in a sterol extract that came from the extracted cells.
xe2x80x9cAlkylxe2x80x9d refers to a branched or unbranched saturated hydrocarbon group. Preferred alkyl groups herein contain 1 to 12 carbon atoms. xe2x80x9cLower alkylxe2x80x9d refers to an alkyl group of 1 to 6, more preferably 1 to 4, carbon atoms.
xe2x80x9cAlkylenexe2x80x9d refers to a molecular fragment that is a saturated branched or unbranched hydrocarbon chain, and includes, for example, ethylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94). xe2x80x9cAlkenylenexe2x80x9d refers to a molecular fragment that is an unsaturated hydrocarbon chain, and includes, for example, ethenylene (xe2x80x94CHxe2x95x90CHxe2x80x94).
xe2x80x9cHydrocarbylxe2x80x9d refers to a branched or unbranched, saturated or unsaturated hydrocarbon group.
xe2x80x9cHaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to fluoro, chloro, bromo or iodo groups. Of the halos, chloro and bromo are generally preferred with chloro generally being the more preferred.
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, xe2x80x9coptionally substituted phenylxe2x80x9d means that the phenyl may or may not be substituted and that the description includes both unsubstituted phenyl and phenyl wherein there is substitution.
As used herein, the term xe2x80x9csterolxe2x80x9d refers to unsaturated hydroxyl group-containing derivatives of a fused, reduced ring system, cyclopenta[a]-phenanthrene, comprising three fused cyclohexane rings (A, B and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D). The exemplary steroid below illustrates the numbering system employed herein in describing the location of groups and substituents. 
Several 5,7-diene containing sterols are candidates for application of the enhanced Diels-Alder reaction of the invention. These include analogs to natural sterols and novel synthetic 5,7-diene containing sterols in addition to natural 5,7-diene-containing sterols. There are several known natural 5,7-diene-containing sterols.
In nature, sterols are derived from acetate in complex biosynthetic cycles that share paths through the production of squalene. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.
U.S. Pat. No. 5,460,949 describes a method for increasing the accumulation of squalene and specific sterols in yeast that comprises increasing the expression level of a structural gene encoding a polypeptide having HMG-CoA reductase activity in a mutant yeast having particular single or double defects in the expression of sterol biosynthetic enzymes.
The accumulation of squalene tends to enhance the production of sterols. In yeast, squalene is converted to squalene epoxide, which is then cyclized to form lanosterol. Lanosterol has two methyl groups at position 4, a methyl group at position 14, a double bond at position 8(9) and an 8 carbon side chain of the formula CH3CH(CH2)2CHxe2x95x90C(CH3)2 bonded to the carbon at position 17. Lanosterol is sequentially demethylated at positions 14 and 4 to form zymosterol (cholesta-8,24-dienol), which is methylated on the side chain at position 17, and ultimately converted to ergosterol (ergosta-5,7,22-trienol), the most abundant sterol of naturally occurring, wild-type yeast. In the double mutant erg5-erg6 yeast utilized illustratively here in Example 1, the 17-position side chain is not methylated because of the erg6 mutation. In addition, the erg5 mutation stops dehydration at C-22 so that the cholesterol-type unsaturation in the C-17 side chain does not occur.
In animals such as mammals, including humans, lanosterol is also an intermediate in the synthesis of 5,7-diene-containing sterols. In one pathway, lanosterol is converted to 24,25-dihydrolanosterol, and then to 4xcex1-methyl-xcex948-cholesterol, 4xcex1-methyl-xcex947-cholesterol, xcex947-cholesterol, 7-dehydrocholesterol (a 5,7-diene) and then to cholesterol. The position 17 side chain is not further alkylated in usual sterol syntheses in animals. Principles of Biochemistry, 6th Ed., Abraham White et al., eds., McGraw-Hill Book Company (New York: 1978), p. 619-630.
In higher plants such as tobacco, cotton, soybean, tomato and alfalfa, the side chain at position 17 is methylated in the formation of obtusifoliol, followed some steps later by a further methylation on that added carbon atom to ultimately form the intermediate xcex947-avenasterol, which is a branch point in the synthesis. In one pathway, stigmasta-5,7,24(28)-trien-3xcex2-ol is formed that leads to stigmasta-5,7-dien-3xcex2-ol and then sitosterol or stigmasterol. In another pathway, xcex947-avenasterol forms stigmasta-7-en-3xcex2-ol, 7-dehydrostigmasterol (a 5,7-diene) and then stigmasterol. See, for example, U.S. Pat. No. 5,589,619, and the citations therein.
The 5,7-diene-containing sterols that are subjected to a Diels-Alder reaction in a process of the invention and that can be isolated and purified according to another process of the invention have the general structural formula: 
wherein R3 is H or R1COxe2x80x94 where R1 is lower alkyl or monocyclic aryl of 5 to 7 carbon atoms, and R4, R5 and R6 are independently selected from the group consisting of H, hydroxyl and lower alkyl. The R3 moiety, if other than H, is often considered a hydroxyl-protecting group. Typical R3 moieties are H, CH3COxe2x80x94 and C6H5COxe2x80x94. If R4, R5 and R6 are other than H or OH, they are preferably methyl or ethyl, more typically methyl. R7 is a 1 to 10 carbon atom hydrocarbyl group that can be straight or branched and saturated or unsaturated.
Structural formulas of preferred 5,7-diene sterols are shown below, and include 7-dehydroepisterol, 7-dehydroavenasterol, ergosterol, ergosta-5,7,22,24-tetraenol, stigmasta-5,7,24(28)-trien-3xcex2-ol, stigmasta-5,7-dien-3xcex2-ol, 7-dehydrositosterol, cholesta-5,7,24-trien-3xcex2-ol and 7-dehydrocholesterol. 
A method of the present invention is particularly useful for isolating and purifying cholesta-5,7,24-triene-3xcex2-ol from a mixture of sterols, as can be obtained from mutant yeast. A mixture of sterols in a mutant yeast extract typically contains the yeast metabolites squalene, lanosterol, 4,4-dimethylzymosterol, and the like in addition to triene. Other dienes present, e.g. other sterols having 5,7-dienes, in the yeast extract solution may also react with the dienophile.
It is also contemplated that other sterol compounds present in the composition from which a particular 5,7-diene-containing sterol is to be isolated contain two or more positions of ethylenic unsaturation, for example in the 8 to 10 carbon atom side chain R7 as well as in the A or B steroid ring. Those diene compounds typically contain ethylenic unsaturation that is unconjugated, whereas the 5,7-double bonds are conjugated in the contemplated 5,7-diene-containing sterols. A contemplated 5,7-diene-containing sterol can also be said to have xcex1,xcex2-ethylenic unsaturation.
In one embodiment of the invention, the mixture of sterols is reacted in the presence of at least a catalytic amount of an exogenously added an unsaturated C12-C24 carboxylic acid with a dienophile having the structural formula Xxe2x80x94Rxe2x95x90Rxe2x80x94Y, wherein the R groups are both N or both C-Q, where the Q groups are H or together form a third bond. Thus, the dienophile in this embodiment has the structure Xxe2x80x94Nxe2x95x90Nxe2x80x94Y, Xxe2x80x94(CQ)xe2x95x90(CQ)xe2x80x94Y, or Xxe2x80x94Cxe2x95x90Cxe2x80x94Y. The substituents X and Y are electron-withdrawing groups that are independently selected from the group consisting of xe2x80x94COOH, xe2x80x94CHO, xe2x80x94NO2, xe2x80x94CN, xe2x80x94COOR8 and xe2x80x94COR8, where R8 is lower alkyl, or X and Y can be linked together to form a xe2x80x94(CO)xe2x80x94Zxe2x80x94(CO)xe2x80x94 bridge. In the latter case; i.e., when X and Y are linked together, the xe2x80x9cZxe2x80x9d linkage is lower alkylene, lower alkenylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, xe2x80x94Sxe2x80x94, or xe2x80x94NR2xe2x80x94 wherein R2 is lower alkyl, H or monocyclic aryl of 5 to 7 carbon atoms with up to 5 ring substituents. Ring substituents are selected from the group consisting of xe2x80x94(CH2)nxe2x80x94NH2, xe2x80x94(CH2)nxe2x80x94COOH, xe2x80x94NO2, halogen and lower alkyl, wherein n is an integer that is zero to 6, inclusive. This type of reaction of a dienophile with a 5,7-diene-containing sterol will sometimes be referred to herein as reaction type (1).
Dienophiles within the aforementioned group are available commercially or can be readily synthesized using starting materials and techniques known to those skilled in the art of synthetic organic chemistry. Examples of particular dienophiles useful herein include the following: 
These dienophiles are available commercially from a number of sources, e.g., from the Aldrich Chemical Company, Milwaukee, Wis. As will be appreciated by those skilled in the art, such dienophiles may also be readily synthesized using conventional techniques. See, e.g., S. W. Moje and P. Beak, J. Org. Chem., 39 (20):2951 (1974), and K. Rufenacht, Helv. Chim. Acta, 51:518 (1968).
In another embodiment, a dienophile precursor is used that is converted in situ into a dienophile with a suitable oxidizing agent. Here, the sterol mixture containing a 5,7-diene-containing sterol is simultaneously reacted in the presence of at least a catalytic amount of a C12-C24 carboxylic acid with the dienophile precursor and with an oxidizing agent effective to oxidize the precursor to an active dienophile. A reaction using a dienophile precursor described below and oxidant along with a mixture containing a 5,7-diene-containing sterol is sometimes referred to herein as reaction type (2).
The dienophile precursor has the structural formula Xxe2x80x94NHxe2x80x94NHxe2x80x94Y wherein X and Y are electron-withdrawing groups that are independently selected from the group consisting of xe2x80x94COOH, xe2x80x94CHO, xe2x80x94NO2, xe2x80x94CN, xe2x80x94COOR8 and xe2x80x94COR8 where R8 is lower alkyl, or X and Y can be linked together to form a xe2x80x94(CO)xe2x80x94Zxe2x80x94(CO)xe2x80x94 bridge. In the exemplary dienophile precursors wherein X and Y are linked together to form a xe2x80x94(CO)xe2x80x94Zxe2x80x94(CO)xe2x80x94 bridge, the xe2x80x9cZxe2x80x9d linkage is lower alkylene, lower alkenylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, xe2x80x94Sxe2x80x94, or xe2x80x94NR2xe2x80x94, wherein R2 is H, lower alkyl or monocyclic aryl of 5 to 7 carbon atoms with up to 5 ring substituents. Ring substituents are selected from the group consisting of lower alkyl, halogen, xe2x80x94NO2, xe2x80x94(CH2)nxe2x80x94NH2, and xe2x80x94(CH2)nxe2x80x94COOH, wherein n is an integer that is zero to 6, inclusive.
Preferably, in this embodiment, Z is monocyclic arylene of 5 to 7 carbon atoms substituted with up to 2 substituents that are xe2x80x94(CH2)nxe2x80x94NH2 or xe2x80x94(CH2)nxe2x80x94COOH, wherein n is an integer that is zero to 6, inclusive. Dienophile precursors within the aforementioned group are available commercially or can be readily synthesized using starting materials and techniques known to those skilled in the art of synthetic organic chemistry (see, e.g., H. D. K. Drew and H. H. Hatt, J. Chem. Soc. 16 (1937)). Examples of particular dienophile precursors useful herein (again, such compounds are available commercially, or can be readily synthesized) include the following: 
In a contemplated 5,7diene-containing sterol production process of the invention, one or more unsaturated C12-C24 carboxylic acids such as linolenic acid, linoleic acid, and oleic acid are exogenously added to the Diels-Alder reaction solutions. Technical grade oleic acid is inexpensive and the impurities present in that commercially available grade, linoleic and linolenic acids, are also active ingredients in a reaction according to the invention. Technical grade oleic acid is a most preferred unsaturated C12-C24 carboxylic acid in a process of the invention.
The unsaturated acid is useful in trace quantities, for example about 0.02 weight percent of the reaction solution. However, its usefulness is not diminished by its presence in a larger quantity. Preferably, the lower amounts are used, about 0.05 to about 5 weight percent, most preferably about 0.1 to about 0.5 weight percent.
Unsaturated acids are present in sterol extracts from yeast fermentations in varying amounts. Typically, the variation is a result of the cellular extraction process. Such extraction often removes most of the unsaturated acids normally present in a cell lysate.
The addition of acid to bring down the pH of a caustic saponification/extraction solution can change the relative amounts of endogenous unsaturated acids that are soluble in the organic phase when protonated versus the ionized form of the endogenous unsaturated acids that are less soluble in the organic phase and more soluble in the aqueous phase of an extraction. The organic phase is the sterol extract. Not only the pH, but also the temperature of the water used to wash the organic phase, affects the amount of endogenous unsaturated acids that remain in the sterol extract. Thus, the amount of endogenous unsaturated acids present in a Diels-Alder reaction may be less than 0.02 weight percent, resulting in a low Diels-Alder adduct yield.
Lowering the pH of the saponification reaction before or during extraction is inadvisable from a practical standpoint for several reasons. The first is safety. The addition of a concentrated acid, such as sulfuric acid, to the caustic solution is an exothermic reaction that results in hot spots, and the splattering and splashing of the caustic and also may lead to structural problems with the reactor. A second reason is charring or the formation of black material in the solution. A third reason is gelling of the solution and soap formation. The latter two points lead to problems handling and successfully separating the 5,7-dienes from the solution. In sum, the best practice is not to add acid to the saponification reaction before or during extraction.
Saponification and extraction of the sterol from the yeast can be carried out using a very wide variety of solvents as may be appreciated by one with skill in the art. The current state of the art is to use hexane or heptane, preferably heptane for extracting the saponified 5,7-diene-containing sterol to make the sterol extract.
In a process according to the invention, the addition of exogenous unsaturated C12-C24 carboxylic acid provides the presence of a minimum amount of unsaturated acid in the Diels-Alder reaction solution. The enhancement of Diels-Alder reaction of the available 5,7-diene-containing sterols by the exogenously provided unsaturated acid thus helps to ensure that a high Diels-Alder adduct yield is obtained and that a low yield is not a result of the removal of xe2x80x9ctoo muchxe2x80x9d endogenous unsaturated acid.
Exemplary unsaturated C12-C24 carboxylic acids useful herein are illustrated below. Use of a C18 unsaturated fatty acid is preferred, with technical grade oleic acid being particularly preferred. 
Exemplary compounds that are not within the scope of the phrase xe2x80x9cunsaturated C12-C24 carboxylic acidsxe2x80x9d include oleyl alcohol, an unsaturated alcohol, and stearic acid, a C18 saturated fatty acid, shown below. 
Any oxidizing agent capable of oxidizing the dienophile precursor to an active dienophile can be used, with the exception of oxidizing agents that interfere with the formation of the Diels-Alder adduct or that interact detrimentally in some other way with any of the sterols in the sterol mixture. Exemplary oxidizing agents include sodium hypochlorite, potassium peroxymonosulfate, lead tetraacetate, iodosobenzene diacetate, N-bromosuccinimide, and t-butyl hypochlorite. Preferably, aqueous sodium hypochlorite is used.
Either of the aforementioned reactions; i.e., reaction of the sterol mixture with a dienophile having the structure Xxe2x80x94Rxe2x95x90Rxe2x80x94Y (Reaction 1), or with a dienophile formed in situ from a dienophile precursor of the structure Xxe2x80x94NHxe2x80x94NHxe2x80x94Y and an oxidizing agent (Reaction 2), results in the formation of a Diels-Alder adduct. These reactions are illustrated in the following schemes: 
Both types of reactions are carried out in an inert atmosphere, in a non-reactive solvent. Polar organic solvents, for example ethyl acetate, methyl propionate and ethyl butyrate, are useful for keeping the sterols dissolved. Many polar organic solvents are known and selection is a matter of choice and there are several factors to consider. Halogenated solvents, such as chloromethane, could be used but are less preferred because of safety and handling considerations in an industrial setting. Lower boiling solvents are preferred for their ease in removal. Mixed solvent systems are also useful in a process of the invention. For example, in a contemplated semisynthetic process of making cholesta-5,7,24-trien-3-ol, the heptane used to make the sterol extract from the saponification reaction is not completely removed, so the reaction solution for the Diels-Alder addition step contains heptane in addition to the polar organic solvent.
With Reaction 2, it is preferred that the oxidizing agent be added gradually to a solution of the steroid and the dienophile precursor in the selected solvent. Reaction 2 is carried out at a temperature of zero degrees C to about 25xc2x0 C., preferably at room temperature (about 20xc2x0 C.). At least about 15 minutes, preferably at least 1 hour, should be allowed for Reaction 2 to occur.
After preparation of the Diels-Alder adduct using either Reaction 1 or Reaction 2, the adduct is removed from the reaction mixture and regenerated to provide the 5,7-diene-containing steroid in isolated form. Removal of the adduct from the reaction mixture is preferably done chromatographically, using, for example, a silica gel column that preferentially binds the Diels-Alder adduct.
The chemical and physical properties of the Diels-Alder adduct can be varied by manipulating the substituents present on the dienophile as well as by varying R3. For example, basic properties can be imparted to the Diels-Alder adduct by the use of a dienophile containing a basic substituent, e.g., xe2x80x94(CH2)nxe2x80x94NH2 where n is an integer that is zero to 6, inclusive, or the like. The adduct is then a basic molecule and separable from the sterol mixture using acid extraction. Similarly, acidic properties can be imparted to the Diels-Alder adduct by the use of a dienophile containing an acid substituent, e.g., xe2x80x94(CH2)nxe2x80x94COOH where n is an integer that is zero to 6, inclusive, or the like. The adduct is then an acidic molecule and separable from the sterol mixture using basic extraction.
Also, as alluded to above, after preparation of the Diels-Alder adduct, the moiety present at R3 can be converted to a functionality that imparts desirable crystallization and/or precipitation properties. For example, a hydroxyl group present at position 3 can be readily converted to a benzoate species, which in turn can make the adduct more crystalline and more readily separable from the sterol mixture.
Regeneration of the 5,7-diene-containing steroid, e.g. from a protection or purification step utilizing Diels-Alder addition, is then accomplished by treatment of the adduct with a reducing agent such as lithium aluminum hydride (xe2x80x9cLAHxe2x80x9d), diisobutyl aluminum hydride (xe2x80x9cDiBALxe2x80x9d), Red-Al(copyright) (a solution of sodium bis(2-methoxy-ethoxy)aluminum hydride in toluene, available from the Aldrich Chemical Company, Inc., Milwaukee, Wis.), or the like. Lithium aluminum hydride is particularly preferred. The reaction proceeds initially at a low temperature; i.e., 10xc2x0 C. or lower (again, as can be obtained by an ice/water bath), followed by, after at least about 30 minutes, warming to at least about 50xc2x0 C. for at least several minutes. Excess reducing agent and any salts or derivatives thereof are then removed, e.g., by hydrolysis plus filtration through Celite(trademark) or the like. Evaporation of the reaction mixture provides the 5,7-diene-containing steroid.
Purification of the regenerated 5,7-diene-containing steroid can then be carried out using any of a number of techniques that are readily appreciated by those of ordinary skill in the art. For example, purification can be effected via recrystallization e.g., using methanol, ethanol, or the like, or using precipitation or chromatographic techniques.
As recited in U.S. Pat. No. 5,391,777, citing its parent, U.S. patent application Ser. No. 07/869,328, now abandoned, where the 5,7-diene-containing steroid is cholesta-5,7,24-triene-3xcex2-ol, the isolated, purified material can be used to prepare cholesta-5,7-diene-3xcex2,25-diol. Cholesta-5,7-diene-3xcex2,25-diol is a biologically important hydroxylated pro-vitamin D3 metabolite that can be converted by sunlight or other well-established photochemical methods to 25-hydroxy vitamin D3. Such vitamin D3 derivatives are useful in a number of contexts, e.g., in topical pharmaceutical formulations (for the treatment of skin disorders or the like), in oral vitamin compositions, and as livestock feed additives.
In a variation on the above-described reactions, chemical conversion of one or more sites on the 5,7-diene-containing steroid can be effected while the molecule is protected in the form of the Diels-Alder adduct. For example, the xcex9424 double bond can be converted to a 24-amino-25-hydroxyl species, a 24,25-dihydroxyl species, or the like. Also, the xe2x80x9cAxe2x80x9d ring of the steroid can be oxidized when the compound is in adduct form. Examples of chemical conversions that can be carried out on the Diels-Alder adduct are described in patent application Ser. No. 07/869,328, noted previously.